Electrophoresis is a well developed chemical analysis technique. A review reference on this subject is Chapter 9 of Chromatography--Fundamentals and Applications of Chromatographic and Electrophoretic Methods, Part A: Fundamentals and Techniques, edited by E. Heftmann, Elsevier Scientific Publishing Company, 1983, herein fully incorporated by reference. Capillary electrophoresis (CE) is an important advance in electrophoresis which was pioneered by Jorgenson and Lukacs as reported in Analytical Chemistry 1298 (1981) and in 222 Science 266 (1983), each of which are herein fully incorporated by reference. Since a small diameter capillary is used in CE, a relatively high applied voltage can be used without generating problematic thermal gradients in the capillary. The efficiency of separation in CE is a function of, among other things, the applied voltage. The efficiency of CE is relatively high, e.g., in excess of 400,000 theoretical plates.
The following is a description of a typical CE experiment. A 50-100 micrometer internal diameter silica capillary tube is filled with a suitable conducting buffer. The outlet end of the capillary is immersed in a reservoir containing the buffer and an electrode. A sample containing fluorescent ions of interest is introduced into the inlet end of the capillary and then the inlet end of the capillary is placed into another reservoir containing the buffer and another electrode. A voltage of 30,000 volts is impressed between the electrodes. A fluorescence detector is positioned near the outlet end of the capillary to detect the ions of interest.
The movement of the sample ions of interest is controlled by two factors: (1) electrophoretic migration; and (2) electroosmotic flow. Electrophoretic migration is the migration of the ions of interest towards the oppositely charged electrode under the influence of the electric field. Electroosmotic flow is bulk flow of the buffer in the capillary when the inside surface of the capillary which is in contact with the buffer comprises fixed charge sites which in turn have corresponding mobile counter-ions in the buffer. An unmodified silica capillary surface comprises silanol (Si--OH) groups that are negatively charged (Si--O--) when the pH of the buffer is greater than about 2, and positively charged (Si--OH.sub.2 +) when the pH of the buffer is less than about 2.
When the surface is negatively charged, then the corresponding mobile counter-ions of the negatively charged surface, e.g., sodium ions (Na+), migrate under the influence of the electric field and in the process drag the bulk solvent with them. Thus, the direction of the electroosmotic flow is from the positive to the negative electrode when the surface is negatively charged.
When the surface is positively charged, then the corresponding mobile counter-ions of the positively charged surface, e.g., biphosphate ions (HPO.sub.4.sup.-2), migrate under the influence of the electric field and in the process drag the bulk solvent with them. Thus the direction of the electroosmotic flow is from the negative to the positive electrode when the surface is positively charged. A positively charged surface can also be obtained, e.g., by adsorbing hydrophobic cations onto the inside surface of the capillary.
When the surface is not charged, then there is no electroosmotic flow. Thus, depending on the charge (positive or negative) of the ions of interest, the nature and extent of capillary surface charging and the polarity of the applied voltage, electroosmosis can augment, counteract or even override the electrophoretic migration. Since sample components to be determined must travel from the inlet end of the capillary to the detector which is located near the outlet end of the capillary, it is essential that they move in the desired direction.
Waters Chromatography Division of Millipore of Milford, Mass. and Dionex Corporation of Sunnyvale, Calif. are the leading domestic manufacturers of CE instruments for the analysis of common ions. The Waters instrument utilizes indirect photometric detection, see for example Jandik et al., LC GC magazine, September 1991 issue, beginning on page 634, herein fully incorporated by reference. The Dionex system uses a photometric detector or a fluorescence detector, see for example the Dionex advertisement in LC GC magazine, September 1991 issue, on page 639, herein fully incorporated by reference. At the present time, the preferred detection method in CE for the determination of common ions is indirect photometric detection.
Even though CE has many advantages, it also has several characteristics that need improvement. For example, the concentration detection limit of CE could be improved.